This invention is an improvement on currently established procedures for the detection of differences in DNA sequences. The detection of differences in DNA sequences is a desirable and necessary procedure in the following exemplary areas; detection and diagnosis of alleles responsible for genetic diseases in humans and other species; detection and diagnosis of DNA sequences associated or linked to genes that may or may not be involved in disease in humans and other species; detection and diagnosis of neoplasms and the effects of therapy on neoplasms; detection of and distinction between different pathogens (e.g., viruses, bacteria and fungi); determining the purity of animal strains and pedigrees; distinguishing and identifying different human and animal samples in forensic medicine.
Frequently the DNA sequence difference to be detected is a single DNA base substitution (point mutation). DNA is normally composed of various combinations of four bases termed Adenine (A); Thymidine (T); Cytosine (C) and Guanosine (G). Thus, an example of a DNA sequence may be ATCGCGATCGT. A point mutation may be the substitution of any of the three bases not normally found at a single position for a base that is normally found at that position. For example, the transmutation of a DNA sequence ATCGCGATCGT to ATCGGGATCGT is a point mutation at the underlined position.
Although point mutations may not account for the majority of differences between randomly selected DNA sequences, they do account for many differences between DNA sequences that are responsible for polymorphisms and "disease" related DNAs.
DNAs that differ only by point mutation are very difficult to distinguish by current technologies. Procedures for detecting point mutations fall into two main categories: (1) procedures which detect point mutations when the precise DNA sequence change can be anticipated; (2) procedures which "scan" for point mutations where the precise nature of the individual DNA gene change is not known. The present invention will work in either situation.
Prior to the present invention, point mutations where there is some knowledge of the DNA sequences differences between the normal and variant DNA have been detected by:
(1) Restriction fragment length polymorphisms (D. Botstein, et al. Am. J. Hum. Genet., 32:314-331 (1980)) or (2) Allele specific oligonucleotide (ASO) probing (G. Angelini, P.N.A.S. (USA), 83:4489-4493 (1986).
In the restriction fragment length polymorphism procedure, restriction endonucleases are used to cut the DNA into various chain lengths which can be measured. In allele specific oligonucleotide probing, single base mismatches are determined by thermodynamic differences. The annealing conditions are set such that perfectly paired strands anneal and non-perfectly paired strands do not anneal.
The polymerase chain reaction (PCR) exemplified by U.S. Pat. Nos. 4,683,202 and 4,683,195 is used to amplify specific DNA sequences, however, PCR does not, by itself, provide a method to detect single base mutations. The PCR may be used in conjunction with other techniques such as the present invention to detect point mutations and other DNA sequence differences.
The current invention, competitive oligonucleotide priming (COP), distinguishes closely related DNA sequences by comparing competitive annealing of two or more DNA sequences closely matched to the DNA sequence of interest. The COP procedure has some similarity to the Allele specific oligonucleotide probing procedure and to the polymerase chain reaction procedure, however, neither ASO probing or PCR amplification procedures utilize the unique competitive annealing assay of the present invention to detect specific sequences differing by a single base.
The COP procedure of the present invention has the advantages of simplicity and speed. Furthermore, no filter for hybridization is needed, it can be used with solid supports and the whole procedure can be automated for decreased cost. It provides a method to solve a long felt need to improve and simplify the detection of single base changes in DNA sequences.